Be careful not to disturb the pellets and handle cells in minimum light from this point forward.
11. Resuspend the cells in 200μL of PBS.
12. Prepare cells for fluorescence microscopy or flow cytometry
analysis as described in Subheadings 3.5 or 3.6, respectively
(see Note 10).
3.4 Two-Step
Labeling: Copper-Free
SPAAC and Tetrazine
“Click” Reactions
This procedure is applicable to metabolic labeling experiments with
6-TreAz, O-TCO-TMM, O-AzTMM-C10, and other SPAAC- or
tetrazine-capable trehalose analogs. This procedure uses a final
probe concentration of 50μM, although this can be adjusted as
needed.
1. Follow steps 1–7 in Subheading 3.1.
2. Add 2μL of 5 mM 6-TreAz, O-TCO-TMM, O-AzTMM-C10
(stocks prepared as described in Subheadings 2.1 and 2.2), or
other SPAAC- or tetrazine-capable trehalose analog (or vehicle
control) to 198μL bacteria. Mix the well contents.
3. Continue through steps 8–13 in Subheading 3.1.
4. Optionally fix the cells as described in step 4 of Subheading 3.2
(see Note 10).
5. Resuspend the cells in 147μL PBS.
6. Add secondary reagent:
(a) For SPAAC: For cells labeled with 6-TreAz or
O-AzTMM-C10, add 3μL of 1 mM Cy3 DBCO (stock
prepared as described in Subheading 2.5) or other
cyclooctyne-conjugated reagent.
(b) For tetrazine ligation: For cells labeled with
O-TCO-TMM, add 3μL of 1 mM Cy3 tetrazine (stock
prepared as described in Subheading 2.5) or other
tetrazine-conjugated reagent.
7. Gently pipette up and down to mix reagents.
8. Cover the plate with aluminum foil and incubate in the dark for
30 min at room temperature.
9. Pellet cells by centrifugation and wash three times as described
in steps 10–13 of Subheading 3.1 to remove excess reagent. Be
careful not to disturb the pellets and handle cells in minimum
light from this point forward.
10. Resuspend the cells in 200μL PBS.
11. Prepare cells for fluorescence microscopy or flow cytometry
analysis as described in Subheadings 3.5 or 3.6, respectively
(see Note 10).
Trehalose Probe Labeling
393
11. Resuspend the cells in 200μL of PBS.
12. Prepare cells for fluorescence microscopy or flow cytometry
analysis as described in Subheadings 3.5 or 3.6, respectively
(see Note 10).
3.4 Two-Step
Labeling: Copper-Free
SPAAC and Tetrazine
“Click” Reactions
This procedure is applicable to metabolic labeling experiments with
6-TreAz, O-TCO-TMM, O-AzTMM-C10, and other SPAAC- or
tetrazine-capable trehalose analogs. This procedure uses a final
probe concentration of 50μM, although this can be adjusted as
needed.
1. Follow steps 1–7 in Subheading 3.1.
2. Add 2μL of 5 mM 6-TreAz, O-TCO-TMM, O-AzTMM-C10
(stocks prepared as described in Subheadings 2.1 and 2.2), or
other SPAAC- or tetrazine-capable trehalose analog (or vehicle
control) to 198μL bacteria. Mix the well contents.
3. Continue through steps 8–13 in Subheading 3.1.
4. Optionally fix the cells as described in step 4 of Subheading 3.2
(see Note 10).
5. Resuspend the cells in 147μL PBS.
6. Add secondary reagent:
(a) For SPAAC: For cells labeled with 6-TreAz or
O-AzTMM-C10, add 3μL of 1 mM Cy3 DBCO (stock
prepared as described in Subheading 2.5) or other
cyclooctyne-conjugated reagent.
(b) For tetrazine ligation: For cells labeled with
O-TCO-TMM, add 3μL of 1 mM Cy3 tetrazine (stock
prepared as described in Subheading 2.5) or other
tetrazine-conjugated reagent.
7. Gently pipette up and down to mix reagents.
8. Cover the plate with aluminum foil and incubate in the dark for
30 min at room temperature.
9. Pellet cells by centrifugation and wash three times as described
in steps 10–13 of Subheading 3.1 to remove excess reagent. Be
careful not to disturb the pellets and handle cells in minimum
light from this point forward.
10. Resuspend the cells in 200μL PBS.
11. Prepare cells for fluorescence microscopy or flow cytometry
analysis as described in Subheadings 3.5 or 3.6, respectively
(see Note 10).
Trehalose Probe Labeling
393